PROJECT SUMMARY Traumatic brain injury (TBI) is a leading cause of mortality and long-term disability worldwide. Over 1.7 million Americans suffer a TBI annually and up to 2% of the population currently lives with the long-term neurological consequences of a previous TBI, placing a $76.5 billion annual economic burden on society. Preventative measures reduce injury incidence and/or severity, yet one-third of hospitalized TBI patients die from injuries that are secondary to the initial trauma. Cerebral edema is a life-threatening neurological complication that promotes elevated ICP and leads to clinical deterioration in the hours and days after a TBI. Unfortunately, neurosurgical approaches to control elevated ICP are limited and efficacious medical therapies to control cerebral edema are lacking, presenting a critical barrier to improving patient prognoses after TBI. The objective of this proposal is to test the overarching hypothesis that generation of neutrophil extracellular traps (NETs) initiates a detrimental cascade that culminates in neurological deterioration after TBI. Specific Aim 1 will test the hypothesis that TLR4 activation mediates NET formation after TBI. Proposed mechanistic studies will demonstrate a key regulatory role for activation of the TLR4 signaling pathway in the formation of NETs after TBI. Specific Aim 2 will test the hypothesis that peptidylarginine deiminase 4 (PAD4) promotes cerebral edema after TBI. Proposed mechanistic studies will use genetic and pharmacological approaches to implicate PAD4, a mediator of TLR4-induced histone hypercitrullination in human and mouse neutrophils, in NET formation and neurological injury after TBI. Specific Aim 3 will test the hypothesis that degradation of NETs improves neurovascular function after TBI. Proof of concept studies will demonstrate that targeted degradation of NETs prevents cerebral microthrombus formation, leading to improved cerebral recirculation and reduced edema after TBI. These findings will provide the rationale for clinical repurposing of recombinant human DNase I (rhDNase1), a safe, FDA-approved therapeutic in widespread clinical use for non-neurological diseases, in the management of acute TBI patients. Expected outcomes of the proposed research include the identification of NETs as critical initiators of acute cerebrovascular dysfunction after TBI. In addition to providing a mechanistic explanation for the deleterious effects of neutrophils after acute injury, our studies will establish a critical framework for the development of targeted therapies to improve TBI outcomes. .